Gastrodin protects retinal ganglion cells through inhibiting microglial-mediated neuroinflammation in an acute ocular hypertension model

AIM：To investigate the neuroprotective effect of gastrodin on retinal ganglion cells（RGCs）in an acute ocular hypertension（AOH）rat model and to identify its possible mechanism.METHODS：AOH rat model was performed in a randomly selected eye by anterior chamber perfusion and either received an intraperitoneal injection with various concentrations of gastrodin or normal saline.After 2wk,the rats were sacrificed.Fluoro Gold was used to label survival RGCs.Immunostaining with anti-Iba1 in the retinal flat mounts to calculate the microglia density in the ganglion cell layer（GCL）.Changes in microglial cytokines,tumour necrosis factor-alpha（TNF-α）and inducible NO synthase（i NOS）were examined with Western blot and reverse transcriptionquantitative polymerase chain reaction.Expression levels of total and phosphorylated p38 mitogen activated protein kinase（MAPK）were determined by Western blot.RESULTS：Results showed that AOH induced significant loss of RGCs and severe microglia activation in the GCL.Besides,AOH increased the phosphorylation of p38 MAPK and promoted the release of microglial cytokines in the retinas.Intraperitoneal injection with dose-dependent gastrodin significantly reduced the loss of RGCs and inhibited retinal microglia activation,accompanied with the decreased expression levels of microglial cytokines and p38 MAPK phosphorylation.CONCLUSION：Gastrodin exerts a neuroprotective effect on RGCs in an acute glaucoma animal model viainhibiting microglia activation and microglial-mediated neuroinflammation.The finding demonstrates the potential application of gastrodin in the neuroprotective therapy of acute glaucoma and other retinal neurodegenerative diseases characterized by microglia activation and RGCs death.

Expression and role of autophagy related protein p62 and LC3 in the retina in a rat model of acute ocular hypertension

AIM: To investigate the expression and possible role of the autophagy related protein p62 and LC3 in the retina based on a rat model of acute ocular hypertension.METHODS: Fifty rats were randomized into five groups: control group A, B, C, and D. Groups A to D all received normal saline perfusion into the anterior chamber with pressure of 80 mm Hg for one hour, and retina tissue was obtained at 6, 12, 24 and 48 h after perfusion respectively, to investigate the activation of autophagy following ischemiareperfusion. The distribution and semi-quantification of autophagy related protein p62 and LC3 in the retina were detected using immunohistochemistry technique. The expression level of these two proteins was evaluated using Western blot.RESULTS: The number of retinal ganglion cells(RGCs) decreased with increasing reperfusion time, and significant reduction in the retinal thickness was observed 48 h after perfusion. In normal adult rats, LC3 protein was mainly expressed in the ganglion cell layer(GCL), and p62 protein was expressed in the nerve fiber layer(NFL), GCL, inner plexiform layer(IPL), inner nuclear layer(INL) and outer plexiform layer(OPL). In comparison to the control group, the expression level of LC3-II was higher in all the experimental groups(P<0.05), with the peak expression at 12 h after reperfusion. Additionally, the expression level of p62 was higher in all the experimental groups than the control(P<0.05, except for group A), with the peak level occurred 24 h after reperfusion. CONCLUSION: Both p62 and LC3 show low level and uneven expression in the retina of normal adult rats. Acute ocular hypertension can lead to upregulation of LC3-II and p62 expression in the retina. Autophagy flux is damaged 12 h after reperfusion, potentially resulting in further loss of RGCs.

Alpha B-crystallin improved survival of retinal ganglion cells in a rat model of acute ocular hypertension

Increased endogenous αB-crystallin protein levels have been shown to reduce cell apoptosis, although the effects of exogenous aB-crystallin protein remain poorly understood. The present study established an acute ocular hypertension model in the right eye of Sprague-Dawley rats. Fluorogold retrograde tracing and immunofluorescence methods showed that the number of retinal ganglion cells decreased in the right eyes and caspase-3 expression increased following acute ocular hypertension. Intravitreal injection of aB-crystallin in the right eye increased the number of retinal ganglion cells and reduced caspase-3 expression. Results demonstrated that exogenous αB-crystallin protein inhibited caspase-3 expression and improved retinal ganglion cell survival following acute ocular hypertension.

Activation of the ATP-P2X pathway by TRPV4 in acute ocular hypertension

AIM:To measure the expression of transient receptor potential cation channel subfamily V member 4(TRPV4)in the rat cornea and determine whether it is related to adenosine triphosphate(ATP)generation in a rat model of acute ocular hypertension(AOH).METHODS:Immunofluorescence staining of TRPV4,P2X2 receptor,P2X3 receptor,and(33-tubulin in rat corneal longitudinal sections and paved was performed to clearly display histological structures.Rat models of AOH and agonist/antagonist-treated groups were established and corneal ATP was measured using an ATP assay.The independent t-test and simple linear correlation model were adopted for statistical analyses.RESULTS:Immunofluorescence staining of rat cornea sections revealed that epithelial and endothelial membranes showed strong immunoreactivity for TRPV4 and P2X2 receptor and coexpression with(33-tubulin in the rat corneal epithelial layer.Corneal ATP was significantly higher in the AOH rat model than in the control(P<0.05)and apparently lower after pretreatment by applying eyedrops of TRPV4 antagonist RN1734 with 30-40 mm Hg intraocular pressure(IOP;P<0.05).A simple linear regression model showed a positive correlation between rat corneal ATP and IOP values(R^(2)=0.996,P=0.0134)from the normal IOP(113 mm Hg)to 40 mm Hg.At 10-40min after anterior chamber injection of GSK1016790A(0.01 mL,50 nmol/L in 0.9%NaCl),corneal ATP was significantly higher than in the control group(P<0.05),which peaked at 1Omin.The ATP concentration of the normal epithelium was higher than that of the endothelium in the AOH rat model and after anterior chamber injection of GSK1016790A(P<0.05).CONCLUSION:The ATP concentration in the AOH rat cornea is increased by TRPV4 activation.

Protective effects of human umbilical cord mesenchymal stem cells on retinal ganglion cells in mice with acute ocular hypertension

AIM:To observe the protective effect of human umbilical cord mesenchymal stem cells(huc MSCs)on retinal ganglion cel s(RGCs)injury in mice with acute ocular hypertension(AOH).METHODS:Fifty-six adult male C57 BL/6 mice were randomly divided into four groups:normal group,AOH group,huc MSCs group,normal saline(NS)group.Left eye of mice was induced by 90 mm Hg intraocular pressure for 1 h to establish AOH model.huc MSCs 1×105/μL,1μL or NS 1μL was injected into the vitreous body the next day.CMDil fluorescent dye was used to label the 3 rd generation of huc MSCs,for tracing the cells in the vitreous cavity of mice.Seven days after the model established,hematoxylin-eosin(HE)staining was used to observe the thickness of the inner retina layer in four groups.Numbers and loss rate of RGCs were evaluated by counting Brn-3 a positive cells stained by immunofluorescencein.RESULTS:On the 7 th day after AOH established,labeled huc MSCs were found in the vitreous cavity.HE staining showed that the thickness of retinal inner layer in AOH group was significantly lower than that in normal group and huc MSCs group(P<0.05),same as that in NS group(P>0.05).Compared with AOH group,the RGCs in normal group was significantly higher;RGCs number increased in huc MSCs group and the loss rate was lower(P<0.05).Injection of NS had no protective effect on RGCs.CONCLUSION:In AOH mouse model,vitreous injection of hucMSCs have shown a protection for RGCs.

A Study of Histology and Enzymatic Histochemistry on Rabbit's Retina in Acute Ocular Hypertension

The changes of activities of enzymes relating to energy metabolism in rabbit's retina in acute ocular hypertension were observed. The activities of succinate dehydrogenase and adenosine triphosphatase were found to be reduced, while the activities of the lactatic dehydrognease and glucose-6-phosphatase increased. The results revealed the metabolic disturbance of energy in retina after acute ocular hypertension might be the underlying factors relating to the defects of the functions and structures of the...

Minocycline inhibits the production of the precursor form of nerve growth factor by retinal microglial cells

A rat model of acute ocular hypertension was established by enhancing the perfusion of balanced salt solution in the anterior chamber of the right eye. Minocycline （90 mg/kg） was administered intraperitoneally into rats immediately after the operation for 3 consecutive days. Immunofluorescence, western blot assay and PCR detection revealed that the expression of the precursor form of nerve growth factor, nerve growth factor and the p75 neurotrophin receptor, and the mRNA expression of nerve growth factor and the p75 neurotrophin receptor, increased after acute ocular hypertension. The number of double-labeled CD11B- and precursor form of nerve growth factor-positive cells, glial fibrillary acidic protein- and p75 neurotrophin receptor-positive cells glial fibrillary acidic protein- and caspase-3-positive cells in the retina markedly increased after acute ocular hypertension. The above-described expression decreased after minocycline treatment. These results suggested that minocycline inhibited the increased expression of the precursor form of nerve growth factor in microglia, the p75 neurotrophin receptor in astroglia, and protected cells from apoptosis.